PROJECT SUMMARY/ ABSTRACT Short tissue preservation times impose numerous constraints on transplantation by contributing to organ shortage, exacerbating ischemic injury and graft rejection, and diminishing the length and quality of life for transplant recipients. For vascular composite allograft (VCA) the current standard of preservation for clinical transplantation, of a few hours of hypothermic static storage in UW solution, on ice, deprives VCA of the desired quality and limits VCA use due to the small time window between procurement and transplantation and hindered donor-recipient matching. Therefore, this preservation proposal to the NIAID?s Division of Transplantation focuses on the VCA due to (i) The vast and urgent need for VCA in reconstructive transplantation, and (b) The fact that VCA is a favorable initial system to develop long-term preservation solutions for organ systems in general, from a technical perspective (evidence suggests extremities are extra cold tolerant vs. internal vital organs) as well as from safety and regulatory perspectives (less life threatening risks vs. vital organ transplants). In this Phase 2 study we will continue to develop a Nature-inspired, non-toxic solution to dramatically extend preservation times of limbs and other VCA to over 5 days, while seeking to be the first to achieve organ preservation via a controlled, partially frozen state at high subzero temperatures (-5 to -30C), integrated with machine perfusion and programmed metabolic depression. This unexplored, highly promising methodology is based on the best strategies employed by freeze-tolerant and hibernating animals in nature, augmented with complementary strategies developed using recent scientific understanding and bioengineering principles. Importantly, we will build on the successful demonstration of feasibility in Phase 1 and the results that Sylvatica and MGH/Harvard have in collaboration demonstrated with regard to high subzero partial freezing preservation: (1) 120h storage, at -20C, of simple in vitro VCA model with viabilities above the 80% target, (2) cryopreservation in a simple blood vessel model with good viability and normal morphology after at least 3 days, and (3) successful initial scale-up in whole livers for 3 days. Therefore, the objective of this Phase 2 proposal is to demonstrate, using animal and human models of limb preservation, prolonged cryopreservation of VCA of 5 days or longer, with good functional outcome post storage and recovery. Across five specific aims, we will first employ simple, yet rich cellular and vascular models of VCA for cryostatis cocktail and protocol optimization, and initial scaling up, with the central goal of enabling high subzero preservation, while actively suppressing metabolism and enhancing stress tolerance. Best cryostasis protocols will be subsequently validated using, first a model of rat forelimb preservation and assessment through sub-and normothermic machine perfusion, and then, orthotopic allotransplantation, with comprehensive characterization of upper extremity functional and behavioral recovery. Following, the best learnings from the animal model preservation/transplantation will be used for the proof of concept for high subzero partial freezing of human fingers and hands, with full pseudo transplantation quality assessment.